Kaoru Otsu

An abortive apoptotic pathway induced by singlet oxygen is due to the suppression of caspase activation

Singlet oxygen causes the cytotoxic process of tumour cells in photodynamic therapy. The mechanism by which singlet oxygen damages cells is, however, not fully understood. To address this issue, we synthesized and used two types of endoperoxides, MNPE (1-methylnaphthalene-4-propionate endoperoxide) and NDPE (naphthalene-1,4-dipropionate endoperoxide), that generate defined amounts of singlet oxygen at 37 degrees C with similar half lives. MNPE, which is more hydrophobic than NDPE, induced the release of cytochrome c from mitochondria into the cytosol and exhibited cytotoxicity, but NDPE did not. RBL cells, a rat basophil leukaemia-derived line, that overexpress phospholipid hydroperoxide glutathione peroxidase in mitochondria were found to be highly resistant to the cytotoxic effect of MNPE. MNPE treatment induced much less DNA ladder formation and nuclear fragmentation in cells than etoposide treatment, even though these treatments induced a similar extent of cellular damage. Singlet oxygen inhibited caspase 9 and 3 activities directly and also suppressed the activation of the caspase cascade. Collectively, these data suggest that singlet oxygen triggers an apoptotic pathway by releasing cytochrome c from mitochondria via the peroxidation of mitochondrial components and results in cell death that is different from typical apoptosis, because of the abortive apoptotic pathway caused by impaired caspase activation.

Accumulation of manganese superoxide dismutase under metal-depleted conditions: proposed role for zinc ions in cellular redox balance

A diet low in copper results in increased levels of MnSOD (manganese superoxide dismutase), a critical antioxidative enzyme conferring protection against oxidative stress, in rat liver mitochondria. The mechanism for this was investigated using cultured HepG2 cells, a human hepatocellular carcinoma-derived line. MnSOD activity increased 5-7-fold during incubation in a medium supplemented with metal-depleted fetal bovine serum, with a corresponding elevation of its mRNA levels. Metal depletion also decreased CuZnSOD and glutathione peroxidase levels to approx. 70-80% of baseline. When zinc ions were added to the medium at micromolar levels, MnSOD accumulation was suppressed; however, copper ions had essentially no effect on MnSOD expression. Since the intracellular redox status was shifted to a more oxidized state by metal depletion, we examined the DNA-binding activity of NF-kappaB (nuclear factor-kappaB), an oxidative stress-sensitive transactivating factor that plays a primary role in MnSOD induction. A gel shift assay indicated that the DNA-binding activity of NF-kappaB was increased in cells maintained in metal-depleted culture, suggesting the involvement of the transactivating function of NF-kappaB in this induction. This was further supported by the observation that curcumin suppressed both the DNA-binding activity of NF-kappaB and the induction of MnSOD mRNA in cells cultivated under metal-depleted conditions. These results suggest that the level of zinc, rather than copper, is a critical regulatory factor in MnSOD expression. It is possible that a deficiency of zinc in the low-copper diet may be primarily involved in MnSOD induction.

Interleukin-6 down-regulates expressions of the aldolase B and albumin genes through a pathway involving the activation of tyrosine kinase

Interleukin-6 plays a key role in mediating acute-phase protein synthesis in hepatocytes. However, the mechanism of how interleukin-6 regulates aldolase B and albumin syntheses in hepatocytes is not completely understood. In this study, using primary cultured rat hepatocytes, we have shown that interleukin-6 down-regulates expressions of the aldolase B and albumin genes in a dose- and time-dependent manner. We examined whether the decrease in aldolase B and albumin mRNA expressions by interleukin-6 reflected transcriptional down-regulation or stability of the mRNA. Actinomycin D and cycloheximide did not affect the interleukin-6-mediated decrease in the expressions of both genes. These results suggest that the decreased expressions of both genes induced by interleukin-6 is controlled at the transcriptional level, and that it is due neither to increased degradation of mRNA nor to synthesis of new proteins. Protein kinases play a fundamental role in the intracellular signal transduction. To examine the interleukin-6 signal pathway(s) leading to the decrease of aldolase B and albumin mRNA expressions, we tested various kinds of protein kinase inhibitors in this system. Herbimycin A, an inhibitor of tyrosine kinase(s), prevented the decrease in the expression of aldolase B and albumin mRNAs by interleukin-6. H-7, an inhibitor of protein kinase C, prevented the decrease in the expression of albumin mRNA by interleukin-6, but did not induce recovery of that of aldolase B mRNA. These results suggest that a tyrosine kinase(s) or a herbimycin A-sensitive kinase(s) constitutes a common pathway for interleukin-6-mediated reduction of aldolase B and albumin mRNA expressions and that distinct pathways exist for the modes of expression of the two mRNAs.

Differential regulation of liver-specific and ubiquitously-expressed genes in primary rat hepatocytes by the extracellular matrix.

Primary rat hepatocytes were cultured with an extracellular matrix (ECM) overlay, in order to investigate the effect of an ECM on gene expression in hepatocytes. When hepatocytes, isolated by the collagenase-perfusion method, were cultured on type I collagen-coated dishes, the mRNA levels of liver-specific genes (aldolase B, tyrosine aminotransferase and albumin) decreased continuously, while those of ubiquitously-expressed genes (glyceraldehyde 3-phosphate dehydrogenase and β-actin) increased. When a dilute ECM derived from the Engelbreth-Holm-Swarm mouse sarcoma (an EHS gel) was added to the above hepatocytes 3 days after plating, the mRNA levels of liver-specific genes increased, while those of ubiquitously-expressed genes decreased. The effects of a rat liver biomatrix (a physiological ECM for rat hepatocytes) on gene expression in primary hepatocytes were similar to those of the EHS gel. A nuclear run-on assay, and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole or actinomycin D treatments revealed that the transcriptional rates of liver-specific genes were enhanced by the EHS gel overlay, while the apparent stability of the corresponding mRNAs were unchanged. In contrast, the transcriptional rates of ubiquitously-expressed genes were not greatly affected by an EHS gel overlay, while the apparent stability of their mRNAs were decreased. These data suggest that the ECM plays an important role in the maintenance of the differentiated characteristics of primary hepatocytes by inducing the transcription of liver-specific genes and, also, by destabilizing the mRNAs of ubiquitously-expressed genes.

Impaired activation of caspase cascade during cell death induced by newly synthesized singlet oxygen generator, 1-buthylnaphthalene-4-propionate endoperoxide.

Endoperoxides of naphthalene derivatives generate singlet oxygen under physiological conditions. Here we have synthesized a new endoperoxide of a naphthalene derivative, 1‐buthylnaphthalene‐4‐propionate endoperoxide (BNPE), and studied its cytotoxic properties on HepG2 and HaCaT cells. BNPE induced cell death at much lower concentration than 1‐methylnaphthalene‐4‐propionate endoperoxide (MNPE) and naphthalene dipropionate endoperoxide (NDPE). A positive correlation exists between the amount of endoperoxide incorporated into cells and its cytotoxic ability. The cytotoxic effect of BNPE was attenuated by α‐tocopherol but not by sodium azide. In contrast, the effects of MNPE and NDPE were attenuated by both α‐tocopherol and sodium azide. The caspase cascade in cells treated with endoperoxide was impaired. Caspase activity in a soluble protein fraction were inhibited similarly by the above three endoperoxides. These results suggest an abortive apoptotic pathway due to the suppression of caspase activation is a general feature of cell death induced by singlet oxygen.